Reese Kimberly J, Lin Shu, Verona Raluca I, Schultz Richard M, Bartolomei Marisa S
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, United States of America.
PLoS Genet. 2007 Aug;3(8):e137. doi: 10.1371/journal.pgen.0030137. Epub 2007 Jun 29.
Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division.
印记基因H19的父源抑制由一个差异甲基化区域（DMD）介导，该区域对于H19以及与之连锁且印记相反的Igf2基因的印记至关重要。DMD的父源特异性甲基化如何在早期胚胎全基因组去甲基化阶段得以保留，随后又如何用于调控印记表达，其机制尚不清楚。甲基化CpG结合（MBD）蛋白可能是解释这些DMD如何被识别以沉默该基因座的候选蛋白，因为它们优先结合甲基化DNA并募集具有组蛋白去乙酰化酶活性的抑制复合物。在植入前胚胎中发现了MBD RNA和蛋白，染色质免疫沉淀显示MBD3与H19 DMD结合。为了测试MBD在印记中的作用，采用了两种基于RNA干扰的独立策略在早期小鼠胚胎中耗尽MBD3，结果相同。在经RNA干扰处理的囊胚中，父源H19表达被激活，这支持了以下假设：作为Mi-2/NuRD复合物成员之一的MBD3是抑制父源H19等位基因所必需的。经RNA干扰处理的囊胚中Mi-2/NuRD复合物蛋白MTA-2的水平也降低了，这表明Mi-2/NuRD抑制复合物在H19基因座的父源特异性沉默中发挥作用。此外，当MBD3蛋白被耗尽时，H19 DMD处的DNA甲基化减少。相比之下，其他印记基因在植入前胚胎中的表达和DNA甲基化并未受到干扰。这些结果证明了MBD3在维持印记控制区域DNA甲基化以及沉默父源H19等位基因方面的新作用。最后,耗尽MBD3的植入前胚胎细胞数量减少，这表明MBD3在细胞分裂中发挥作用。
